This project will extend the principal investigator's basic and clinical research on regulation of blood coagulation involving studies of protein-protein and protein-lipid interactions and on translational clinical research studies of venous thrombosis risk factors. Thrombosis is strongly linked to failure to regulate thrombin generation, i.e., to an imbalance in anticoagulant and procoagulant mechanisms. One major goal of this project is to elucidate molecular interactions that are critical for thrombin generation. We hypothesize that molecular mechanisms for regulation of thrombin generation by the blood coagulation pathways are determined by specific amino acids on surfaces of coagulation factors V (fV) and X (fX) and by specific lipids that determine the assembly of the procoagulant prothrombinase complex (fXa:fVa:phospholipid:Ca++). Preliminary data from synthetic peptides that inhibit fVa or fXa functions combined with analysis of fVa and fXa three-dimensional structures enable us to propose specific mutations in fVa and fXa that we speculate will cause loss of functional activities. In specific aims 1-4, the fVa and fXa mutants will be studied using coagulation and prothrombinase assays and fluorescence spectroscopy to identify dysfunctional molecules and to characterize the nature of their dysfunction. Based on preliminary data showing that sphingosine and other long chain alkyl amines are potent anticoagulants, we propose to clarify how sphingosine inhibits thrombin generation by neutralizing the prothrombinase complex activity. A second major goal involves translational thrombosis research. As in the previous period of support, we propose continued studies that build a bridge between plasma dyslipoproteinemia research and blood coagulation research. Based on preliminary data, we propose studies to test the hypothesis that risk of excessive thrombin generation in vivo, manifested as venous thrombosis, is associated with HDL2b deficiency and that this is caused by genetic variations (SNPs) in genes that regulate lipoprotein and HDL metabolism. The proposed studies will increase our insights into the pathophysiology of thrombosis and are likely to improve diagnosis and treatment of thrombosis.